1. Field of The Invention
This invention relates generally to a method for calibrating a flow cytometer or fluorescence microscope in terms of number of binding antibodies as a function of fluorescence lntensity value measured on the flow cytometer or fluorescence microscope, and subsequently measuring a sample to which such antibodies are bindable. The invention also relates to a microbead calibration kit suitable for carrying out such calibration method.
2. Background and Description of the Art
The development of monoclonal antibodies as analytical probes for the detection of cell surface antigen expression combined with the establishment of sophisticated techniques such as flow cytometry have greatly contributed to improve the clinical diagnosis and medical treatment of a variety of immunological diseases. The identification of a particular sub-type of cell, even when present in minuscule numbers (e.g. B cells in malignant lymphomas), has been possible due to the high resolution and sensitivity provided by the development of flow cytometry. The flexibility, sensitivity, and accuracy of flow cytometry not only allows detection of cell populations which occur in small numbers, but also offers the opportunity to quantitate fluorescence intensity and epitope expression at extremely low levels (e.g., 3400 FITC molecules).
Within the past few years, many new functional sub-populations of cell types have been indentified and studied in heterogeneous hematopoietic and lymphoid human cell populations. Recent efforts have been directed toward determining the percentage of each particular sub-population present and the use of this variable as a diagnostic tool to distinguish between normal and malignant cells. It has been shown, for example, that changes in the T4/T8 (helper/supressor) ratio of lymphocytes, as well as total T4 cells, have a high correlation with certain stages in the progression of Acquired immune Deficiency Syndrome (AIDS).
The recognition of the clinical relevance of the availability of antibody binding sites and/or antibody binding site density per cell is at a very early stage of dvelopment. Quantitation of specific antibody binding sites on certain cells will have increasing utility and importance in clinical diagnosis as early indicators of the onset of a particular disease, as well as in monitoring the dosage and toxicity of certain drugs and the efficacy of treatment regimes. Quantitation of the antigenic sites-may also provide a useful index of the progression and/or remission of immune related disease.
Presently, efforts to quantitate cellular epitopes have been significantly limited by the lack of standard reagents that will allow quantitation and accurate interpretation of the fluorescence signals. At best, determination of the number of antibody binding sites per cell is an extremely difficult technique to accomplish and thus, has remained a research methodology. For example, direct radio-binding assays have been used to quantitate the number of monoclonal antibodies to cells in research laboratories. This method is extremely tedious, however, and requires the incorporation of gamma-emitting iodine isotopes which are a biohazard requiring special laboratory facilities and licenses. Moreover, the cost of the methodology is high due to the equipment required and the short half-life of the iodine isotopes. These factors have seriously limited the utilization of this methodology in the clinical laboratory.
Theoretically, the number of antibody binding sites per cell can be assessed using flow cytometry. One methodology would involve calibrating the fluorescence channels of the instrument in terms of absolute numbers of molecules of fluorescent dye and also determining the average number of fluorescence dye molecules conjugated to the antibody molecule, usually referred to as the F/P ratio. In this manner, the average number of antibodies binding to cells could be calculated by determining the total number of fluorescent dye molecules associated with a particular antibody-labeled cell (via the instrument calibration) and dividing by the F/P ratio. The underlying assumption is that antibody binding at saturation is monovalent.
Unfortunately, this simplistic approach does not take into account the environmental conditions and instrument parameters that affect the measurement of fluorescence intensity of fluorochromes, such as pH of the suspension medium, quenching resulting from molecule to molecule proximity, changes in extinction coefficient due to binding and the selection of barrier filters and gains. Consequently, to determine absolute numbers of fluorochrome molecules present, "corrected fluorescence emission spectra" must be obtained based on many correction factors and quantum efficiencies. Such determinations and calculations are also tedious and extremely time consuming. This is perhaps the primary reason that antibody quantitation by means of fluorescence is not clinically practical and has remained a research methodology.
Recognizing such limitations, a series of products called Quantitative Fluorescent Microbead Standards Kits.TM., commercially available from Flow Cytometry Standards Corporation (Research Triangle Park, N.C.) have been developed which provide for the quantitation of fluorescence intensity in terms of equivalent soluble fluorescent molecules, rather than absolute numbers of fluorescent molecules. This approach focuses on the relative intensity of the fluorescence signal of a sample compared to soluble solutions of the specific dye under controlled environmental conditions. The underlying premise is that their fluorescence intensities will maintain a direct quantitative relationship, when the excitation and emission spectra of the sample and solution match, and both are measured under the same environmental conditions and instrument settings. Moreover, a standard particle with spectra matching the solution of the dye, will hold the same quantitative relationship with other particles, e.g., fluorescent antibody-labeled cells, as long as the fluorescent spectra of the sample match that of the standard particle and the dye solution. This criterion renders the fluorescence intensity measurements independent of the instrument settings, filters, and environmental factors, as long as the standard particles and the samples are measured under similar conditions. This method eliminates the need for the above mentioned correction factors since the number of molecules of fluorochrome present are no longer the determinant, but rather the fluorescence intensity of the particular sample relative to a soluble solution of the fluorochrome of a known concentration.
Recently, a special microbead capable of binding a calibrated number of antibodies has become available under the tradename Simply Cellular.TM. Microbeads (Flow Cytometry Standards Corporation, Research Triangle Park, N.C.). Briefly, with this product the number of antibody binding sites in a cell can be determined by first calibrating the fluorescence detector of the flow cytometer with the aforementioned quantitative microbead standards (Quantitative Fluorescent Microbead Standards Kit.TM.) and constructing a calibration plot of instrument response (i.e., channel number) as a function of equivalent soluble fluorescent molecules. Subsequently, an aliquot of the Simply Cellular.TM. Microbeads is saturated with the fluorescent monoclonal antibody to be studied using direct or indirect immunofluorescence methods and its fluorescence intensity determined against the calibration plot. The "effective fluorescence F/P ratio" is calculated by dividing the fluorescence intensity in terms of equivalent soluble fluorescent molecules by the number of antibody molecules binding to the Simply Cellular.TM. Microbeads (this information is provided by the manufacturer). Cell samples to be examined are also saturated with the same fluorescent monoclonal antibody under the same environmental conditions used with the microbeads and are analyzed with the flow cytometer using identical instrument settings. The fluorescence intensity of the cell samples is determined against the calibration plot and the average number of antibodies binding to the cell calculated by dividing the fluorescence intensity of the antibody-cell complex by the previously calculated "effective fluorescence F/P ratio".
This method of quantitating antibody binding has been found to be reliable. The utilization of this method in the clinical laboratory, however, will be limited by the number of steps involved in the procedure and the requirement of a specific set of Quantitative Fluorescent Microbead Standards that matches the excitation and emission spectra of the fluorochrome (e.g., fluorescein, phycoerythrin, Texas red, allophycocyanine, etc.) conjugated to the monoclonal antibody to be evaluated.
It is therefore an object of the present invention to provide a simple and readily conducted method for calibrating a flow cytometer or a fluorescence microscope directly in terms of number of binding antibodies as a function of fluorescence intensity value measured on the flow cytometer or fluorescence microscope.
It is another object of the invention to provide a calibration method of the type described in the preceding paragraph, which provides a direct relationship between instrument response and numbers of binding antibodies, independent of the conjugated fluorochrome. It is a further object of the invention to provide a microbead calibration kit for carrying out the above-referenced method of calibrating a flow cytometer or fluorescence microscope. Other objects and advantages will be more fully apparent ensuing disclosure and appended claims.